Expression cassette encoding a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and herbicide-tolerant plants containing it

ABSTRACT

The present invention relates to a novel expression cassette comprising a nucleic acid sequence encoding an EPSPS. In particular, the present invention relates to a novel expression cassette comprising, in the direction of transcription, functionally linked to one another, a promoter regulatory sequence which is functional in plant cells or plants, a nucleic acid sequence encoding an EPSPS and a terminator sequence which is functional in plant cells or plants, characterized in that the promoter regulatory sequence is a nucleic acid sequence chosen from the promoter regulatory sequences of the CsVMV (Cassava Vein Mosaic Virus) plant virus.

RELATED APPLICATIONS

This application is a continuation application of application Ser. No.12/569,068, filed Sep. 29, 2009, which is a divisional application ofapplication Ser. No. 10/538,438 filed Jan. 3, 2006, which is a nationalstage application (under 35 U.S.C. 371) of PCT/EP2003/015008 filed Dec.10, 2003, which claims benefit to French application 0215695 filed Dec.12, 2002. The entire contents of each of these applications are herebyincorporated by reference herein in their entirety.

The present invention relates to a novel expression cassette comprisinga nucleic acid sequence encoding a 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) and to its use for obtaining plants resistant toherbicides which inhibit this enzyme, in particular herbicides of thephosphonic acid family, in particular of the N-phophonomethylglycinefamily.

STATE OF THE ART

One of the main problems in agriculture lies in controlling thedevelopment of undesirable self-propagating plants, or weeds, in areaswhere crops are grown. The development of weeds leads to a weakening ofthe crop plants and to a decrease in the yields from growing them. Inorder to combat these undesirable plants, herbicides are used, generallyby spraying onto the crops.

Many types of herbicide exist, in particular selective herbicides whichact only on a group of particular plants without affecting the cropplants. The disadvantage of selective herbicides is that their spectrumof activity is generally restricted, which requires the use of otherselective herbicides with different spectra of activity in order toeffectively control weeds. The solution to this disadvantage lies in theuse of total herbicides capable of acting on all plants. Totalherbicides are often herbicides for which the target is enzymes involvedin the vital metabolic pathways of plants, which gives them theadvantage of having a broad spectrum of activity on plants of distantphylogenetic origins. However, such herbicides also have the majordisadvantage, when they are applied to crops in order to eliminateweeds, of also acting on the crop plants. This disadvantage may beovercome by using crop plants tolerant to said herbicides. Such plantsare generally obtained by genetic engineering, by introducing into theirgenome a gene encoding an enzyme resistant to said herbicide so thatthey overexpress said enzyme in their tissues.

EPSPS is a plastid enzyme involved in the shikimate biosyntheticpathway, leading to the synthesis of aromatic amino acids. EPSPS isknown to be the target enzyme for herbicides of the family of phosphonicacids of the phophonomethylglycine type.5-enolpyruvylshikimate-3-phosphate synthase (EPSPS)-inhibitingherbicides are well known as being highly effective foliar herbicides.The most well known herbicide of this herbicide class is glyphosate[N-(phosphonomethyl)glycine]. Sulfonate or fosametine are also known.Glyphosate is characterized by a lack of selectivity for crop speciesand is, consequently, generally used under conditions in which there isno need for selectivity, for example as a total herbicide.

In order to overcome the problem of selectivity of glyphosate, plantstolerant to this herbicide have been developed by transformation of saidplants with a gene encoding a glyphosate-tolerant EPSPS enzyme. Genesencoding glyphosate-tolerant EPSPS enzymes are in particular describedin patent application EP 0837944. In particular, glyphosate-tolerantmaize and soybean are sold respectively under the trade marksRoundup-Ready Corn™ and Roundup-Ready Soybean™. In this way, glyphosatecan be applied to crops without affecting the crop plants which havebeen made tolerant thereto.

The success of this strategy is essentially based on the quality and thequantity of expression of the enzyme in the tissues of the plantintended to be made tolerant. These parameters of quality and quantityof expression are controlled by the regulatory elements introduced intothe expression cassette with the nucleic acid sequence encoding saidEPSPS enzyme. The regulatory elements essential to an expressioncassette are the promoter regulatory sequence and the terminatorregulatory sequence. The expression cassettes can also contain a signalpeptide or a transit peptide, and also a transcription activator elementor enhancer. However, the regulatory element which contributes most tothe quality and the quantity of expression of a protein encoded by anucleic acid sequence in an expression cassette is the promoter.Identification of the promoter suitable for expression of a givenprotein also depends largely on the nature of said protein, and inparticular on the desired quantity and quality of expression of saidprotein. Associated with a given promotor is a quantity of expression ofthe product encoded by the nucleic acid sequence which it controls, andalso a quality, in particular spatiotemporal quality, of thisexpression. In addition, some promoters are constitutive and othersinducible.

An important characteristic for a promoter used in an expressioncassette intended for the expression of an EPSPS enzyme in a plant isthat it should allow a quantitative expression sufficient to confertolerance to glyphosate on all the tissues of the plant which may beaffected by this herbicide.

The technical problem of the present invention consists in obtaining anexpression cassette in which the promoter is particularly suitable forthe quantitative and qualitative expression of an EPSPS enzyme intransformed plants, said expression cassette then conferring effectivetolerance on said plants, with respect to a herbicide which inhibitsthis enzyme, in particular a herbicide of the phophonomethylglycinefamily, in particular glyphosate.

Promoters which allow a high level of expression are generally promotersof highly expressed proteins. Among the most commonly used promoterssatisfying these criteria, mention may be made, by way of example, ofbacterial promoters, such as that of the octopine synthase gene or thatof the nopaline synthase gene, viral promoters, such as that of the genecontrolling transcription of cauliflower mosaic virus 35S or 19S RNAs(Odell et al., 1985, Nature, 313, 810-812), or promoters of the cassavavein mosaic virus (as described in patent application WO 97/48819).Among the promoters of plant origin, mention will be made of thepromoter of the ribulose-biscarboxylase/oxygenase (RuBisCO) smallsubunit gene, the promoter of a histone gene described in application EP0 507 698, or the promoter of a rice actin gene (U.S. Pat. No.5,641,876).

Other promoters are expressed specifically in the cells of certaintissues. Such promoters are generally promoters which regulate theexpression of proteins involved in the function of a particular tissueor organ. In plants, root-specific promoters, such as, for example, thatdescribed in patent application WO 00/29594, flower-specific promoters,such as those described in patent applications WO 98/22593, WO 99/15679or WO 99/43818, and fruit-specific promoters, in particularseed-specific promoters such as those described in patent applicationsWO 91/13993, WO 92/17580, WO 98/45460, WO 98/45461, or WO 99/16890, areknown.

DESCRIPTION

The present invention relates to a novel expression cassette comprising,in the direction of transcription, functionally linked to one another, apromoter regulatory sequence which is functional in plant cells orplants, a nucleic acid sequence encoding5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) and a terminatorsequence which is functional in plant cells or plants, characterized inthat the promoter regulatory sequence is a nucleic acid sequence chosenfrom the promoter regulatory sequences of the CsVMV (Cassava Vein MosaicVirus) plant virus.

The expression “functionally linked to one another” means that saidelements of the chimeric gene are linked to one another in such a waythat their function is coordinated and allows expression of the codingsequence. By way of example, a promoter is functionally linked to acoding sequence when it is capable of ensuring expression of said codingsequence. The construction of a chimeric gene according to the inventionand the assembly of its various elements can be carried out usingtechniques well known to those skilled in the art, in particular thosedescribed in Sambrook et al. (1989, Molecular Cloning: A LaboratoryManual, Nolan C. ed., New York: Cold Spring Harbor Laboratory Press).The expression “functional in plant cells and plants” is intended tomean capable of functioning in plant cells and plants.

CsVMV promoter regulatory sequences are described in patent applicationWO 97/48819 (the content of which is incorporated herein by way ofreference), in particular the promoter regulatory sequence comprisingone of the nucleotide sequences represented by one of the sequenceidentifiers SEQ ID Nos. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15 or 16 of patent application WO 97/48819, more particularly thenucleic acid sequence represented by the sequence identifier SEQ ID NO 3of patent application WO 97/48819.

According to a first preferred embodiment of the invention, the promoterregulatory sequence of the expression cassette comprises, in thedirection of transcription, the nucleic acid sequences X, Y and Z asdefined respectively by SEQ ID NOS 1, 2 and 3 of the present patentapplication.

According to the present invention, a preferred promoter regulatorysequence for the expression cassette is represented by SEQ ID NO 4 ofthe present patent application.

According to a second embodiment of the invention, the promoterregulatory sequence of the expression cassette comprises, in thedirection of transcription, the nucleic acid sequences X, Y, Y and Z asdefined above. The promoter regulatory sequence comprising theduplication of the nucleic acid sequence Y will be referred to as doubleCsVMV. Preferably, the nucleic acid sequence of the double CsVMV isrepresented by SEQ ID NO 5 of the present application.

The present invention also relates to the sequences capable ofhybridizing selectively with the nucleic acid sequences above, thesequences homologous to the sequences above, and the functionalfragments of said sequences.

According to the present invention, the term “nucleic acid sequence” isintended to mean a nucleotide or polynucleotide sequence which may be ofthe DNA or RNA type, preferably of the DNA type, in particulardouble-stranded.

According to the invention, the expression “sequence capable ofhybridizing selectively” is intended to mean the sequences whichhybridize with the sequences above at a level significantly greater thanthe background noise. The background noise may be related to thehybridization of other DNA sequences present, in particular other cDNAspresent in a cDNA library. The level of the signal generated by theinteraction between the sequence capable of hybridizing selectively andthe sequences defined by the SEQ IDs above according to the invention isgenerally 10 times, preferably 100 times, more intense than that of theinteraction of the other DNA sequences generating the background noise.The level of interaction can be measured, for example, by labeling theprobe with radioactive elements, such as ³²P. Selective hybridization isgenerally obtained using very stringent medium conditions (for example0.03 M NaCl and 0.03 M sodium citrate at approximately 50° C.-60° C.).The hybridization can of course be carried out according to the usualmethods of the state of the art (in particular Sambrook & al., 1989,Molecular Cloning: A Libratory Manual).

According to the invention, the term “homologue” is intended to mean anucleic acid fragment exhibiting one or more sequence modificationsrelative to the nucleotide sequence encoding the fusion protein of theinvention. These modifications may be obtained according to the usualmutation techniques, or else in choosing the synthetic oligonucleotidesused in the preparation of said sequence by hybridization. With regardto the multiple combinations of nucleic acids which may lead to theexpression of a same amino acid, the differences between the referencesequence according to the invention and the corresponding homologue maybe considerable. Advantageously, the degree of homology will be at least70% relative to the reference sequence, preferably at least 80%, morepreferably at least 90%. These modifications are generally andpreferably neutral, i.e. they do not affect the primary sequence of thefusion protein.

The methods for measuring and identifying homologies between nucleicacid sequences are well known to those skilled in the art. Use may, forexample, be made of the PILEUP or BLAST programs (in particular Altschul& al., 1993, J. Mol. Evol. 36: 290-300; Altschul & al., 1990, J. Mol.Biol. 215: 403-10).

The methods for measuring and identifying homologies betweenpolypeptides or proteins are also known to those skilled in the art. Usemay, for example, be made of the UWGCG package and the BESTFITT programfor calculating homologies (Deverexu & al., 1984, Nucleic Acid Res. 12,387-395).

According to the invention, the term “fragments” is intended to meanfragments of the DNA sequences according to the invention, i.e. thesequences above for which parts have been deleted but which conserve thefunction of said sequences.

According to the invention, the term “EPSPS” is intended to mean anynative or mutated 5-enolpyruvylshikimate-3-phosphate synthase enzyme,the enzymatic activity of which consists in synthesizing5-O-(1-carboxyvinyl)-3-phosphoshikimate from phosphoenolpyruvate (PEP)and 3-phosphoshikimate (E.C. 2.5.1.19; Morell et al., 1967, J. Biol.Chem. 242, 82-90). In particular, said EPSPS enzyme may originate fromany type of organism. An EPSPS enzyme according to the invention alsohas the property of being tolerant with respect to herbicides of thephophonomethylglycine family, in particular with respect to glyphosate.

Sequences encoding EPSPSs which are naturally tolerant, or are used assuch, with respect to herbicides of the phophonomethylglycine family, inparticular glyphosate, are known. By way of example, mention may be madeof the sequence of the AroA gene of the bacterium Salmonella typhimurium(Comai et al., 1983, Science 221, 370-371), the sequence of the CP4 geneof the bacterium Agrobacterium sp. (WO 92/04449), or the sequences ofthe genes encoding Petunia EPSPS (Shah et al., 1986, Science 233,478-481), Tomato EPSPS (Gasser et al., 1988, J. Biol. Chem. 263,4280-4289), or Eleusine EPSPS (WO 01/66704).

Sequences encoding EPSPSs made tolerant to glyphosate by mutation arealso known. By way of example, mention may be made of the sequences ofthe genes encoding mutated EPSPSs of bacterial origin (Stalker et al.,1985, J. Biol. Chem. 260 (8), 4724-4728), or of plant origin (EP0293358; Ruff et al., 1991, Plant Physiol. 96(S), Abstract 592; WO91/04323; WO 92/06201; EP 0837944). A sequence of a gene encoding amutated plant EPSPS which is preferred according to the invention isthat encoding the maize EPSPS described in patent application EP0837944, comprising a first mutation replacing the threonine amino acidat position 102 with isoleucine, and a second mutation replacing theproline amino acid at position 106 with serine. Due to the strongsequence homology between EPSPSs, and more particularly between plantEPSPSs, a rice EPSPS carrying the same mutations have also beendescribed in patent applications WO 00/66746 and WO 00/66747. Ingeneral, any EPSPS, and the genes encoding them, carrying thethreonine/isoleucine and proline/serine mutations described above,whatever the relative position of these amino acids with respect topositions 102 and 106 of maize EPSPS, can be used in the presentinvention. To apply this principle, those skilled in the art will bereadily able to find the two amino acids to be mutated in any EPSPSsequence by using standard techniques of sequence alignment.

According to a preferred embodiment of the invention, the nucleic acidsequence encoding an EPSPS included in the expression cassette is asequence encoding an EPSPS which has been mutated at the amino acidscorresponding to the threonine at position 102 and to the proline atposition 106, said positions being relative with respect to the maizeEPSPS sequence.

According to another preferred embodiment of the invention, the nucleicacid sequence encoding an EPSPS included in the expression cassette is asequence encoding a mutated EPSPS comprising an isoleucine at position102 and a serine at position 106, said positions being relative withrespect to the maize EPSPS sequence.

According to another preferred embodiment of the invention, the nucleicacid sequence encoding an EPSPS included in the expression cassette is asequence encoding maize mutated EPSPS comprising an isoleucine atposition 102 and a serine at position 106.

Among the terminator sequences which can be used in the expressioncassette according to the present invention, mention may be made, by wayof example, of the nos terminator sequence of the gene encodingAgrobacterium tumefaciens nopaline synthase (Bevan et al., 1983, NucleicAcids Res. 11 (2), 369-385), or the terminator sequence of a histonegene as described in application EP 0 633 317.

The expression cassette according to the invention may also comprise asubcellular addressing sequence encoding a signal peptide or transitpeptide. Such a sequence, located upstream or downstream of the nucleicacid sequence encoding the EPSPS, makes it possible to direct said EPSPSspecifically into a cellular compartment of the host organism. Forexample, the expression cassette may comprise a sequence encoding asignal peptide or a transit peptide for directing the EPSPS to aparticular compartment of the cytoplasm, such as the mitochondria, theplants, the endoplasmic reticulum or the vacuoles.

The role of such sequences is in particular described in issue 38 of therevue Plant Molecular Biology (1998), devoted in large part to thetransport of proteins into the various compartments of the plant cell(Sorting of proteins to vacuoles in plant cells pp 127-144; the nuclearpore complex pp 145-162; protein translocation into and across thechloroplastic enveloppe membranes pp 91-207; multiple pathways for thetargeting of thylakoid proteins in chloroplasts pp 209-221;mitochondrial protein import in plants pp 311-338).

According to one embodiment, the transit peptide may be a signal forchloroplastic or mitochondrial addressing, which is then cleaved in thechloroplasts or the mitochondria.

The transit peptides may be either single or double transit peptides.The double transit peptides are optionally separated by an intermediatesequence, i.e. they comprise, in the direction of transcription, asequence encoding a transit peptide of a plant gene encoding an enzymewhich is located in plastids, a portion of sequence of the matureN-terminal portion of a plant gene encoding an enzyme which is locatedin plastids, and then a sequence encoding a second transit peptide of aplant gene encoding an enzyme which is located in plastids. Such doubletransit peptides are, for example, described in patent application EP 0508 909.

According to the invention, the expression cassette may also compriseother regulatory sequences, which are located between the promoter andthe coding sequence, such as transcription activators (enhancers), forinstance the transcription activator of the tobacco mosaic virus (TMV)described in application WO 87/07644, of the tobacco etch virus (TEV)described by Carrington & Freed, or of the figwort mosaic virus (U.S.Pat. No. 5,994,521), for example. The expression cassette according tothe invention may also contain introns, in particular introns whichpromote gene expression in monocotyledon plants, such as intron 1 of therice actin gene described in patent application WO 99/34005, or themaize adh1 intron, or in dicotyledon plants, such as the Arabidopsishistone intron (EP 0850311).

The present invention also relates to a cloning an/or expression vectorcomprising an expression cassette according to the invention. The vectoraccording to the invention is of use for transforming a host organism,in particular a plant, and expressing therein an EPSPS. This vector maybe a plasmid, a cosmid, a bacteriophage or a virus. Preferably, thevector for transforming plant cells or plants according to the inventionis a plasmid. In general, the main qualities of this vector should be anability to maintain itself and to self-replicate in the cells of thehost organism, in particular by virtue of the presence of an origin ofreplication, and to express therein an EPSPS. The choice of such avector and also the techniques for inserting therein the expressioncassette according to the invention are widely described in Sambrook etal. (1989, Molecular Cloning: A Laboratory Manual, Nolan C. ed., NewYork: Cold Spring Harbor Laboratory Press) and are part of the generalknowledge of those skilled in the art. Advantageously, the vector usedin the present invention also contains, in addition to the expressioncassette according to the invention, another expression cassettecontaining a selection marker. This selection marker makes it possibleto select the host organisms which have effectively been transformed,i.e. those which have incorporated the vector. According to a particularembodiment of the invention, the host organism to be transformed is aplant. Among the selection markers which can be used, mention may bemade of markers containing genes resistant to antibiotics, such as, forexample, that of the hygromycin phosphotransferase gene (Gritz et al.,1983, Gene 25:179-188), but also markers containing genes for toleranceto herbicides, such as the bar gene (White et al., NAR 18:1062, 1990)for tolerance to bialaphos, the EPSPS gene (EP 0837944) for tolerance toglyphosate or else the HPPD gene (WO 96/38567) for tolerance toisoxazoles. Mention may also be made of genes encoding readilyidentifiable enzymes such as the GUS enzyme, and genes encoding pigmentsor enzymes which regulate the production of pigments in the transformedcells. Such selection marker genes are in particular described in patentapplications WO 91/02071, WO 95/06128, WO 96/38567, and WO 97/04103.

The present invention also relates to plant cells transformed with avector as described above. The term “transformed plant cell” is intendedto mean a plant cell which has incorporated into its genome theexpression cassette according to the invention, and consequentlyproduces an EPSPS. To obtain the transformed plant cells according tothe invention, those skilled in the art may use one of the many knownmethods of transformation. One of these methods consists in bringing theplant cells to be transformed into contact with polyethylene glycol(PEG) and the vectors of the invention (Chang and Cohen, 1979, Mol. Gen.Genet. 168 (1), 111-115; Mercenier and Chassy, 1988, Biochimie 70 (4),503-517). Electroporation is another method, which consists insubjecting the plant cells or tissues to be transformed and the vectorsof the invention to an electric field (Andreason and Evans, 1988,Biotechniques 6 (7), 650-660; Shigekawa and Dower, 1989, Aust. J.Biotechnol. 3 (1), 56-62). Another method consists in directly injectingthe vectors into the plant cells or the plant tissues by microinjection(Gordon and Ruddle, 1985, Gene 33 (2), 121-136). Advantageously, the“biolistic” method may be used. It consists in bombarding plant cells orplant tissues with particles onto which the vectors of the invention areabsorbed (Bruce et al., 1989, Proc. Natl. Acad. Sci. USA 86 (24),9692-9696; Klein et al., 1992, Biotechnology 10 (3), 286-291; U.S. Pat.No. 4,945,050). Preferably, the transformation of the plant cells willbe carried out using bacteria of the genus Agrobacterium, preferably byinfection of the cells or tissues of said plants with A. tumefaciens(Knopf, 1979, Subcell. Biochem. 6, 143-173; Shaw et al., 1983, Gene 23(3):315-330) or A. rhizogenes (Bevan and Chilton, 1982, Annu. Rev.Genet. 16:357-384; Tepfer and Casse-Delbart, 1987, Microbiol. Sci. 4(1), 24-28). Preferably, the transformation of plant cells withAgrobacterium tumefaciens is carried out according to the protocoldescribed by Ishida et al. (1996, Nat. Biotechnol. 14 (6), 745-750).Those skilled in the art will choose the appropriate method according tothe nature of the plant cells to be transformed.

A subject of the present invention is a method for producing plantstolerant to EPSPS-inhibiting herbicides, in particular to herbicides ofthe phophonomethylglycine family, in particular glyphosate. This methodconsists in regenerating transformed plants from the transformed plantcells described above. By this method, the transformed plants accordingto the invention contain an expression cassette according to theinvention in their genome and express an EPSPS in their tissues.

The present invention therefore comprises transformed plants comprisingan expression cassette according to the invention, parts of theseplants, and the descendants of these plants. The expression “part ofthese plants” is intended to mean any organ of these plants, whetheraerial or subterranean. The aerial organs are the stems, the leaves andthe flowers. The subterranean organs are mainly the roots, but they mayalso be tubers. The term “descendants” is intended to mean mainly theseeds containing the embryos derived from the reproduction of theseplants with one another. By extension, the term “descendants” applies toall the seeds formed at each new generation derived from crosses betweena plant and the plant transformed by the method according to theinvention.

A subject of the present invention is therefore transformed plants intothe genome of which there is integrated at least one expression cassetteaccording to the invention in a stable manner.

The plants thus transformed are tolerant to EPSPS-inhibiting herbicides,in particular herbicides of the phophonomethylglycine family, inparticular to glyphosate.

The transformed plants according to the invention also include thetransformed plants derived from growing and/or crossing the plantsabove, and also the seeds of such plants.

Of course, the transformed cells and plants according to the inventionmay comprise, in addition to an expression cassette according to theinvention, at least one other expression cassette containing apolynucleotide encoding a protein of interest. Among the polynucleotidesencoding a protein of interest, mention may be made of polynucleotidesencoding another enzyme for resistance to a herbicide, for example thepolynucleotide encoding the bar enzyme (White et al., NAR 18:1062, 1990)for tolerance to bialaphos, or the polynucleotide encoding the HPPDenzyme (WO 96/38567; WO 99/24585; WO 99/24586) for tolerance toisoxazoles. Mention may also be made of a polynucleotide encoding aninsecticidal toxin, for example a polynucleotide encoding a toxin of thebacterium Bacillus thuringiensis (for example, see International PatentApplication WO 98/40490). Other polynucleotides for resistance todiseases may also be contained in these plants, for example apolynucleotide encoding the oxalate oxydase enzyme as described inpatent application EP 0 531 498 or U.S. Pat. No. 5,866,778, or apolynucleotide encoding an antibacterial and/or antifungal peptide suchas those described in patent applications WO 97/30082, WO 99/24594, WO99/02717, WO 99/53053, and WO 99/91089. Mention may also be made ofpolynucleotides encoding plant agronomic characteristics, in particulara polynucleotide encoding a delta-6 desaturase enzyme as described inU.S. Pat. Nos. 5,552,306 and 5,614,313, and patent applications WO98/46763 and WO 98/46764, or a polynucleotide encoding a serineacetyltransferase (SAT) enzyme as described in patent applications WO00/01833 and WO 00/36127.

The additional expression cassettes may be integrated by means of thevector according to the invention. In this case, the vector comprisesthe expression cassette according to the invention and at least oneexpression cassette encoding another protein of interest.

They may also be integrated by means of at least one other vectorcomprising said additional expression cassette, according to the usualtechniques defined above.

The plants according to the invention may also be obtained by crossingparents, one carrying the expression cassette according to theinvention, the other carrying another expression cassette encoding atleast one other protein of interest.

The transformed plants according to the invention may be monocotyledonsor dicotyledons. Preferably, these plants are plants of agronomicinterest. Advantageously, the monocotyledon plants are wheat, maize orrice, advantageously, the dicotyledon plants are oilseed rape, soybean,tobacco or cotton.

The present invention also relates to a method for protecting cropplants with respect to EPSPS-inhibiting herbicides, in particular toherbicides of the phophonomethylglycine family, in particular toglyphosate, characterized in that said plants are transformed with avector comprising an expression cassette according to the invention.

The present invention also relates to a method for treating the plantsaccording to the invention, characterized in that said plants aretreated with EPSPS-inhibiting herbicide, in particular a herbicide ofthe phophonomethylglycine family, in particular glyphosate.

The present invention also relates to a method for controlling weeds incrops, characterized in that transformed plants comprising an expressioncassette according to the invention are grown, and in that said plantsare treated with an EPSPS-inhibiting herbicide, in particular aherbicide of the phophonomethylglycinse family, in particularglyphosate.

The present invention also relates to a method for growing transformedplants comprising an expression cassette according to the invention,characterized in that it consists in planting seeds of said transformedplants in an area of a field suitable for growing said plants, inapplying to said area of said field an agrochemical composition, withoutsubstantially affecting said transformed seeds or said transformedplants, then in harvesting the plants grown when they have reached thedesired maturity and, optionally in separating the seeds from theharvested plants.

According to the invention, the term “agrochemical composition” isintended to mean any agrochemical composition comprising at least oneactive product having one of the following activities: herbicidal,fungicidal, bactericidal, virucidal or insecticidal.

According to a preferred embodiment of the method of growing accordingto the invention, the agrochemical composition comprises at least oneactive product having at least one herbicidal activity, more preferablyan EPSPS-inhibiting herbicide, in particular a herbicide of thephophonomethylglycine family, in particular glyphosate.

The various aspects of the invention will be understood more clearlyfrom the experimental examples below.

All the methods or operations described below in these examples aregiven by way of example and correspond to a choice made from the variousavailable methods for achieving the same result. This choice has nobearing on the quality of the result and, consequently, any suitablemethod may be used by those skilled in the art to achieve the sameresult. In particular, and unless otherwise specified in the examples,all the recombinant DNA techniques used are carried out according to thestandard protocols described in Sambrook et al. (1989, MolecularCloning: A Laboratory Manual, Second edition, Nolan C. ed., Cold SpringHarbor Laboratory Press, NY), in Sambrook and Russel (2001, Molecularcloning: A laboratory manual, Third edition, Cold Spring HarborLaboratory Press, NY), in Ausubel et al. (1994, Current Protocols inMolecular Biology, Current protocols, USA, Volumes 1 and 2), and inBrown (1998, Molecular Biology LabFax, Second edition, Academic Press,UK). Standard materials and methods for plant molecular biology aredescribed in Croy R. D. D. (1993, Plant Molecular Biology LabFax, BIOSScientific Publications Ltd (UK) and Blackwell Scientific Publications(UK)). Standard materials and methods for PCR (Polymerase ChainReaction) are also described in Dieffenbach and Dveksler (1995, PCRPrimer: A laboratory manual, Cold Spring Harbor Laboratory Press, NY)and in McPherson et al. (2000, PCR—Basics: From background to bench,First edition, Springer Verlag, Germany).

EXAMPLES Example 1 Cloning of the CsVMV Promoter Sequence into theMultiple Cloning Vector pRD 254

The plasmid pILTAB 357 provided by The Scripps Research Institute (LaJolla, Calif., USA) contains the following elements in a pBIN 19 vector(Clontech):

-   -   CsVMV promoter (sequence described by SEQ ID NO 4)    -   multiple cloning site    -   NOS terminator

Three regions have been defined in the CsVMV promoter sequence, calledCsVMV X, CsVMV Y and CsVMV Z.

-   -   CsVMV X: from position 10 to position 227 (SEQ ID NO 1, length        218 bp)    -   CsVMV Y: from position 228 to position 394 (SEQ ID NO 2, length        167 bp)    -   CsVMV Z: from position 397 to position 522 (SEQ ID NO 3, length        126 bp)

In the original sequence of the CsVMV promoter, the X and Y regions areadjacent and the Y and Z regions are separated by the 2 bp sequence AT.

The cloning vector pRD 254 corresponds to the commercial vectorpBlueScript II SK (−) (Clontech) which has undergone mutagenesis so asto replace the unique Sca I site contained in the ApR gene with a Pvu IIsite.

The 532 bp contained between the Hind III and Xba I sites of pILTAB 357were cloned into the cloning vector pRD 254, so as to obtain the plasmidpRD 257.

Example 2 Creation of a CsVMV-EPSPS Expression Cassette

An expression cassette introduced into a plasmid called pSF29 wasdeveloped.

The pSF29 cassette comprises the CsVMV promoter as described by thesequence identifier SEQ ID NO 4, the sequence encoding the optimizedtransit peptide (OTP) as defined in patent application EP 0508909, thesequence encoding maize EPSPS comprising the mutationsthreonine102isoleucine and proline106serine as described in patentapplication EP 0837944, and the nos terminator as described in Bevan etal. (1983, Nucleic Acids Res. 11 (2), 369-385).

Example 3 Integration of the Expression Cassette into an AgrobacteriumT-DNA-Vector

A shuttle plasmid is used to be recombined in the superbinary plasmidpTVK 291 (Jun et al., 1987). Recombination between the unique COS sitespresent on the two plasmids produces a single circular moleculecorresponding to fusion of the two plasmids.

Recombination between pSF29 and the superbinary plasmid pTVK 291 wasobtained by three-parent crossing with DH5 alpha [pSF29], C2110 [pTVK291], and the JC2073 strain (“helper” strain). The resulting plasmid iscalled pSFK29. The strain obtained, C2110 [pSFK29], was selected on LBmedium containing the 3 antibiotics gentamycin, kanamycin and nalidixicacid. The nalidixic acid allows the selection of C2110 against DH5 alphaor JC2073; since C2110 contains a chromosomal resistance to nalidixicacid which cannot be transferred to the other strains during crossing.Combining gentamycin and kanamycin makes it possible to select thebacteria containing pSF29 and pTVK 291. In addition, pSF29 cannotreplicate in C2110, unless it has been recombined with pTVK 291, sinceC2110 contains the origin of replication RK2 carried by pTVK 291, butnot the origin of replication pBR 322 carried by pSF29.

The recombinant plasmid was then transferred into the Agrobacteriumstrain LBA 4404 via a second three-parent cross. The resultant strain,LBA 4404 [pSFK29], was selected on AB medium (selective forAgrobacterium) containing kanamycin and gentamycin.

Example 4 Transformation of Maize by Agrobacterium tumefaciens with aCsVMV-EPSPS Expression Cassette

Transformation of the maize Zea mays by Agrobacterium is carried outaccording to the method described in Ishida, Y. et al., (1996, NatureBiotechnology, 14, 745-750). The disarmed Agrobacterium tumefaciensstrain described in Example 3 is cocultured with immature maize embryos.A selection with 0.88 mM glyphosate is applied to the embryos. Thetransformation events obtained from the resistant calices are thenback-crossed and their descendants are tested for glyphosate tolerance.

Example 5 Tests for Glyphosate Tolerance of the Transformation Events

20 seeds per event obtained were sown in a greenhouse in small pots in arich compost, and a treatment post-emergence at the 3- to 4-leaf stagewas carried out with a dose of glyphosate corresponding to 4 kg/ha (i.e.4 kg of active material per 500 l) using a calibrated treatment tower.The events which survive the treatment are then counted. The resultsshow that 90% of the transformation events containing the CsVMV-EPSPSexpression cassette have plants capable of tolerating a dosecorresponding to 4 kg/ha of glyphosate. A disparity in the number oftolerant plants for each event tested is due to the fact that theseevents are heterozygotes for the trait (the CsVMV-EPSPS expressioncassette) and have one or more integration loci, and that sometransformation events, by virtue of the position of insertion of thecassette, provide better expression of the EPSPS, and therefore bettertolerance to glyphosate.

The invention claimed is:
 1. A promoter regulatory sequencepolynucleotide which is functional in plant cells or plants comprising,in the direction of transcription, the nucleic acid sequences X, Y, Yand Z, wherein (i) X, Y and Z are SEQ ID NO: 1, 2 and 3, respectively;or (ii) wherein X, Y and Z are SEQ ID NO: 1, 2 and 3, respectively, andat least one of the nucleic acid sequences X, Y or Z is a fragment ofsaid nucleic acid sequence X, said nucleic acid sequence Y, or saidnucleic acid sequence Z.
 2. The promoter regulatory sequencepolynucleotide of claim 1, wherein at least one of the nucleic acidsequences X, Y or Z is a fragment of said nucleic acid sequence X, saidnucleic acid sequence Y, or said nucleic acid sequence Z.
 3. Thepromoter regulatory sequence polynucleotide of claim 1, wherein one ofthe two Y nucleic acid sequences is a fragment of the Y nucleic acidsequence.
 4. The promoter regulatory sequence polynucleotide of claim 1,wherein said promoter regulatory sequence polynucleotide is SEQ ID NO:5.
 5. An expression cassette comprising a polynucleotide, saidpolynucleotide comprising, in the direction of transcription,functionally linked to one another, the promoter regulatory sequencepolynucleotide of claim 1, a nucleic acid sequence encoding an herbicidetolerance protein and a terminator regulatory sequence which isfunctional in plant cells or plants.
 6. The expression cassetteaccording to claim 5, wherein the nucleic acid sequence coding anherbicide tolerance protein is a nucleic acid sequence encoding an EPSPSprotein.
 7. The expression cassette according to claim 5, wherein thenucleic acid sequence polynucleotide encoding an EPSPS is a sequencepolynucleotide encoding an EPSPS which has been mutated at the aminoacids corresponding to the threonine at position 102 and to the prolineat position 106, said positions being relative with respect to the maizeEPSPS sequence.
 8. The expression cassette as claimed in claim 7,wherein the nucleic acid sequence encoding an EPSPS is the sequenceencoding a mutated EPSPS comprising an isoleucine at position 102 and aserine at position 106, said positions being relative with respect tothe maize EPSPS sequence.
 9. The expression cassette as claimed in claim8, wherein the nucleic acid sequence encoding a mutated EPSPS is thesequence encoding a maize mutated EPSPS comprising an isoleucine atposition 102 and a serine at position
 106. 10. The expression cassetteas claimed in claim 5, further comprising a double transit peptidecomprising, in the direction of transcription, a sequence encoding atransit peptide of a plant gene encoding an enzyme which is located inplastids, a portion of sequence of the mature N-terminal portion of aplant gene encoding an enzyme which is located in plastids, and then asequence encoding a second transit peptide of a plant gene encoding anenzyme which is located in plastids.
 11. A cloning and/or expressionvector for transforming plant cells or plants, wherein it comprises atleast one expression cassette as claimed in claim
 5. 12. A transformedplant cell, wherein it comprises an expression cassette as claimed inclaim
 5. 13. A transformed plant, wherein it comprises an expressioncassette as claimed in claim
 5. 14. A seed of a transformed plant asclaimed in claim 13, wherein said seed comprises said expressioncassette.
 15. A method for treating plants as claimed in claim 13,wherein said plants are treated with an EPSPS-inhibiting herbicide. 16.The method as claimed in claim 15, wherein said EPSPS-inhibitingherbicide is glyphosate.
 17. A method for controlling weeds in crops,comprising growing transformed plants as claimed in claim 13, andtreating said plants with an EPSPS-inhibiting herbicide.
 18. The methodas claimed in claim 17, wherein said EPSPS-inhibiting herbicide isglyphosate.
 19. A method for growing the transformed plaits plant asclaimed in claim 13, comprising planting seeds of said transformedplants in an area of a field suitable for growing said plants, applyingto said area of said field an agrochemical composition, withoutsubstantially affecting said transformed seeds or said transformedplants, harvesting the plants grown when they have reached the desiredmaturity and, optionally, separating the seeds from the harvestedplants.
 20. The promoter regulatory sequence polynucleotide of claim 1,wherein X, Y and Z are SEQ ID NO: 1, 2 and 3, respectively.